The designed enzymatic synthesis of oligosaccharide-containing molecules has recently gained prominence in the art as greater numbers of glycosyl transferase enzymes have become available to skilled workers. See, for instance, U.S. Pat. Nos. 5,180,674 and 5,278,299. Indeed, two of the transferase enzymes discussed in U.S. Pat. No. 5,180,674 are an N-acetylgalactosamine transferase that transfers an N-acetylgalactosaminyl (GalNAc) group and an N-acetylglucosaminyl transferase that transfers an N-acetylglucosaminyl (GlcNAc) group.
The glycosyl groups GalNAc and GlcNAc are present alone and together in many naturally occurring oligosaccharide-containing compounds. For example, Beyer et al., Adv. Enzymol., 52:23-175 (1981) report eight different types of linkages for GalNAc and seventeen for GlcNAc. Those compound types include not only oligosaccharides, but also glycoproteins and glycopeptides, proteoglycans, glycolipids and gangliosides.
The blood group antigens are exemplary of three important oligosaccharide compounds in which both glycosyl groups are present. For example, the non-reducing terminal groups of the A-blood group saccharides are GalNAc.alpha.1.fwdarw.3(Fuc.alpha.1.fwdarw.2)Gal.beta.-R', where R' is 1.fwdarw.3GlcNAc or 1.fwdarw.3GalNAc. The terminal saccharides of the O-blood group are Fuc.alpha.(1.fwdarw.2)Gal.beta.-R', whereas the B-blood group terminal saccharides are Gal.alpha.(1.fwdarw.3)(Fuc.alpha.1.fwdarw.2)Gal.beta.-R' where each R' of the O- and B-blood groups is as above.
Each of GalNAc and GlcNAc is transferred to a specific acceptor by a transferase enzyme that recognizes the acceptor structure and the donor form of the glycosyl group. That donor form is a sugar nucleotide; i.e., UDP-GlcNAc or UDP-GalNAc.
Because the O-blood group antigen can be converted to the A-blood group antigen, the reactions of GalNAc and its transfer to a Fuc.alpha.(1-2)Gal.beta.-containing acceptor are among the most widely studied glycosyl transfer reactions. Indeed, U.S. Pat. No. 4,569,909 to Seno et al. teaches the use of uridine diphosphate-N-acetylglucosamine 4-epimerase to epimerize UDP-GlcNAc into an equilibrium mixture of UDP-GlcNAc and UDP-GalNAc. That mixture, after boiling to stop enzymic activity and centrifugation to remove the denatured enzyme, provided a "rough" preparation of UDP-GalNAc that was used with an .alpha.-N-acetylgalactosaminyl transferase referred to as "A-transferase" to convert Type O red blood cells into Type A red blood cells.
Seno et al. did not utilize the epimerase and transferase enzymes in the presence of each other. Seno et al. also began each of their reactions with UDP-GlcNAc, a compound that is relatively difficult to prepare and store in large quantity. Seno et al. also had no concept of a regeneration step in which UDP-GalNAc is recycled.
In addition to the blood group antigens, GlcNAc and GalNAc are present in several other important oligosaccharide compounds. For example, GlcNAc is present in both sialyl Lewis.sup.x (SLe.sup.x) and sialyl Lewis.sup.a (SLe.sup.a), both GlcNAc and GalNAc are present in the mucin oligosaccharides, and GalNAc is present as part of the T-antigen.
Thus, although the individual glycosyl transfer reactions of UDP-GalNAc and UDP-GlcNAc have been studied, as has the epimerization of UDP-GlcNAc to UDP-GalNAc and vice versa, the use of one or the other of GalNAc-1-phosphate and GlcNAc-1-phosphate as a starting material for the enzyme-catalyzed glycosyl transfer of the other saccharide to an acceptor, with regeneration of the UDP-glycoside in a one-pot system has not been taught except by the present inventor and colleagues in their recently published paper, Wong et al., Pure & Appl. Chem., 65(4):803-808 (1993). The present invention provides such a system and provides a skilled worker with a UDP-glycosyl donor whose concentration is not controlled by the equilibrium position of the epimerase reaction.